Estimation of D-Arabinose by Gas Chromatography/Mass Spectrometry as Surrogate for Mycobacterial Lipoarabinomannan in Human Urine.
Estimation of D-Arabinose by Gas Chromatography/Mass Spectrometry as Surrogate for Mycobacterial Lipoarabinomannan in Human Urine.
复制标题
DOI：
10.1371/journal.pone.0144088
复制
发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Chatterjee D
中科院分区：
文献类型：
--
作者：
De P;Amin AG;Valli E;Perkins MD;McNeil M;Chatterjee D
在全球范围内，结核病每年都在缓慢下降，据估计，在2000年至2013年期间，通过有效的诊断和治疗挽救了3700万人的生命。目前，诊断依赖于通过一系列痰显微镜、培养和分子检测在临床标本中证实结核分枝杆菌(Mtb)。用于检测尿中结核分枝杆菌脂糖链阿拉伯甘露聚糖(LAM)作为活动性结核病标志物的商业免疫分析侧向流动试剂盒灵敏度较低，特别是在具有免疫能力的个体中，可能是由于分析物丰度较低。我们目前的研究旨在开发一种方法，以定量的方式验证从培养确认的结核病患者获得的人尿样中LAM的存在。在这里，我们描述了一种从尿液中分离LAM并使用气相色谱/质谱仪定量作为LAM代理的D-阿拉伯糖的综合方法。对从储存库获得的298个尿样进行了严格的分析，发现其含有不同数量的LAM当量，范围在~10-40 ng/mL之间。为了进一步证实在样本中检测到的D-阿拉伯糖来自LAM，即结核硬脂酸，在LAM的磷脂酰肌醇末端存在的唯一的10-甲基十五烷酸也在一组样本中进行了分析，证实D-阿拉伯糖确实来自LAM。在144个来自培养阴性结核病嫌疑人的样本中，30个样本显示存在D-阿拉伯糖，这表明分析物的另一个来源，如播散性结核病或来自非结核分枝杆菌。我们的工作证实，LAM存在于培养阳性患者的尿样中，数量很少，但很容易检测到。这项研究进一步证实，尿液中的LAM是活动性结核病的一个强有力的生物标志物。
Globally, tuberculosis is slowly declining each year and it is estimated that 37 million lives were saved between 2000 and 2013 through effective diagnosis and treatment. Currently, diagnosis relies on demonstration of the bacteria, Mycobacterium tuberculosis (Mtb), in clinical specimens by serial sputum microscopy, culture and molecular testing. Commercial immunoassay lateral flow kits developed to detect Mtb lipoglycan lipoarabinomannan (LAM) in urine as a marker of active TB exhibit poor sensitivity, especially in immunocompetent individuals, perhaps due to low abundance of the analyte. Our present study was designed to develop methods to validate the presence of LAM in a quantitative fashion in human urine samples obtained from culture-confirmed TB patients. Herein we describe, a consolidated approach for isolating LAM from the urine and quantifying D-arabinose as a proxy for LAM, using Gas Chromatography/Mass Spectrometry. 298 urine samples obtained from a repository were rigorously analyzed and shown to contain varying amounts of LAM-equivalent ranging between ~10–40 ng/mL. To further substantiate that D-arabinose detected in the samples originated from LAM, tuberculostearic acid, the unique 10-methyloctadecanoic acid present at the phosphatidylinositol end of LAM was also analyzed in a set of samples and found to be present confirming that the D-arabinose was indeed derived from LAM. Among the 144 samples from culture-negative TB suspects, 30 showed presence of D-arabinose suggesting another source of the analyte, such as disseminated TB or from non-tuberculosis mycobacterium. Our work validates that LAM is present in the urine samples of culture-positive patients in small but readily detectable amounts. The study further substantiates LAM in urine as a powerful biomarker for active tuberculosis.